In eukaryotes, the majority of targeted protein degradation is carried out by the ubiquitin-proteasome system (UPS). The UPS regulates myriad cellular processes and maintains cellular homeostasis through degradation of short-lived regulatory proteins, misfolded proteins or misassembled protein complexes. A subset of the UPS is the endoplasmic reticulum-associated degradation pathway (ERAD), which targets proteins in the ER. ERAD has been implicated in over 60 human diseases, highlighting the medical relevance of ERAD basic research. For example, ERAD specifically targets and degrades rate-limiting enzymes in sterol synthesis, a contributing pathway to atherosclerosis and resulting heart disease. Current knowledge of the UPS and ERAD is heavily influenced by genetic and biochemical studies carried out in the model organism Saccharomyces cerevisiae. Two major ERAD ubiquitin ligases exist in this organism. The following proposal focuses on one of them, Doa10, a conserved ERAD transmembrane ubiquitin ligase, which targets a distinct set of regulatory proteins (including Erg1, a key enzyme in sterol synthesis), as well as various misfolded proteins. Doa10 thus serves in both regulatory and quality-control capacities. This broad array of substrates is uncommon for ubiquitin ligases, and the molecular mechanisms underlying ERAD ubiquitin ligase specificity remain largely unknown. To determine the full range of natural Doa10 substrates and the biological processes it regulates, we will employ a novel screen to identify physiological Doa10 substrates. We have created a substrate trap using a chimeric protein of Doa10 fused to high-affinity ubiquitin-binding domains. Trapped substrates will be identified by mass spectrometry and validated using well established functional assays for Doa10. We will subsequently use these substrates and Erg1 to analyze Doa10 substrate specificity. Our initial focus will be on an intrinsically disordered cytoplasmic loop of Doa10 tha is required for substrate degradation and which our preliminary data suggest is a key region controlling substrate specificity. This region will be systematically analyzed for its contribution to substrate binding, ubiquitination and degradation. Deciphering Doa10 substrate specificity will have a broad impact on our understanding general ubiquitin ligase-substrate interactions. Finally, our studies will be expanded to TEB4, the human ortholog of Doa10. We will examine the conservation of substrate specificity and how it impacts sterol synthesis and downstream cholesterol levels.